J Clin Oncol 24: by American Society of Clinical Oncology INTRODUCTION

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1 VOLUME 24 NUMBER 36 DECEMBER JOURNAL OF CLINICAL ONCOLOGY O R I G I N A L R E P O R T Immunohistochemical Detection Using the New Rabbit Monoclonal Antibody SP1 of Estrogen Receptor in Breast Cancer Is Superior to Mouse Monoclonal Antibody 1D5 in Predicting Survival Maggie C.U. Cheang, Diana O. Treaba, Caroline H. Speers, Ivo A. Olivotto, Chris D. Bajdik, Stephen K. Chia, Lynn C. Goldstein, Karen A. Gelmon, David Huntsman, C. Blake Gilks, Torsten O. Nielsen, and Allen M. Gown From the Genetic Pathology Evaluation Centre at Vancouver General Hospital, Prostate Centre, and University of British Columbia; Breast Cancer Outcomes Unit and Population and Preventive Oncology Programs at BC Cancer Agency, Vancouver, British Columbia, Canada; and PhenoPath Laboratories, Seattle, WA. Submitted December 28, 2005; accepted August 9, 2006; published online ahead of print at on November 20, Supported by a Translation Acceleration grant from the Canadian Breast Cancer Research Alliance, an unrestricted educational grant from sanofi aventis, and the National Institute of Health Strategic Partnering to Evaluate Cancer Signatures program. C.D.B., D.H., and T.O.N. are recipients of Scholar Awards from the Michael Smith Foundation for Health Research. Presented in part at the 28th San Antonio Breast Cancer Symposium, December 8-11, 2005, San Antonio, TX. Authors disclosures of potential conflicts of interest and author contributions are found at the end of this article. Address reprint requests to Torsten O. Nielsen, Anatomical Pathology, JP 1401, Vancouver Hospital & Health Sciences Centre, 855 West 12th Ave, Vancouver, BC, V5Z 1M9, Canada; torsten@interchange.ubc.ca by American Society of Clinical Oncology X/06/ /$20.00 DOI: /JCO A B S T R A C T Purpose Estrogen receptor (ER) expression predicts improved breast cancer specific survival and reduced risk of recurrence and is targeted in breast cancer therapy. A high-quality antibody to identify ER-positive patients plays an important role in clinical decision making for women with breast cancer. This study evaluates immunohistochemistry using two anti-er antibodies, a new rabbit monoclonal antibody (SP1) and the mouse monoclonal antibody (1D5), in relation to biochemical ER assay results and clinical data on survival and adjuvant systemic therapy. Patients and Methods A population-based tissue microarray series of 4,150 invasive breast cancers was constructed. All patients had staging, pathology, treatment, and follow-up information. The median follow-up was 12.4 years and the median age at diagnosis 60 years. Survival analysis and log-rank tests were used to evaluate the prognostic value of ER status and correlations with clinical data. Results Among the 4,105 samples interpretable for both antibodies, SP1 detected ER positivity in 69.5% and 1D5 in 63.1% of cases. Both monoclonal antibodies are demonstrated to be good prognostic indictors for breast cancer specific and relapse-free survival. In multivariate analysis, including age, tumor size, grade, and lymphovascular and nodal status, SP1 was a better independent prognostic factor than 1D5. Among patients with discrepant ER results, the 8% of patients who were SP1 positive/1d5 negative showed good outcomes, and the 2% SP1-negative/1D5 positive had poor outcomes. Maintaining the same 92% specificity and 98% positive predictive value, SP1 is 8% more sensitive than 1D5 using biochemical assay as gold standard. Conclusion SP1 represents an improved standard for ER immunohistochemistry assessment in breast cancer. J Clin Oncol 24: by American Society of Clinical Oncology INTRODUCTION Estrogen receptor (ER) may be the best example of a tumor biomarker with an assay that drives therapeutic decision making. 1-4 In the 1990s, immunohistochemical(ihc) evaluationoferlargelysupplanteddextrancoated charcoal (DCC) ligand-binding assay because it is more economical and yields concurrent histopathologic correlation. Several anti-er antibodies are used clinically; two mouse monoclonals (6F11 and 1D5) havebeencomparedinclinicalstudiesandwereshown to have similar sensitivities. 5,6 Studies have also compared 1D5 with a new rabbit monoclonal, SP1, 7,8 which has eight-fold higher affinity. 8 Our preliminary studies found that SP1 is more sensitive than 1D5 for detecting ER expression in breast cancer, in both a duplicate-redundancy 431-sample TMA, and in 121 whole sections of clinical materials from multiple institutions 9 (Tables A1 and A2, online only). We present the first population-based series comparing the IHC detection of ER by antibodies 1D5 and SP1, and results from DCC in patients with long-term clinical followup, to investigate the prognostic values of these assays in breast cancer. PATIENTS AND METHODS Study Population The study cohort is 4,150 female patients with newly diagnosed, invasive breast cancer in British Columbia, whose tumor specimens were tested by a central (ER) 5637

2 Cheang et al laboratory at Vancouver Hospital between 1986 and The DCC protocol is as published and reproduced on our web site ( papers/er.php). Median follow-up was 12.4 years and age at diagnosis 60 years. All patients had been referred to the British Columbia Cancer Agency and have staging, pathology, treatment, and follow-up information. 13,14 During the study era, 75% of breast cancer patients in the province were referred; nonreferred patients were generally elderly or treated by mastectomy without indications for adjuvant therapy. 15 Abstracted clinical information includes age; histology; grade; tumor size; number of involved axillary nodes; lymphatic or vascular invasion (LVI); ER status by the DCC method 11 ; type of local and initial adjuvant systemic therapy (AST); and dates of diagnosis, first local, regional, or distant recurrence, and death. A subset of these patients was included in a recent population-based study validating the prognostic model ADJUVANT! 16 Table A3 (online only) summarizes cohort characteristics. The study was approved by the Clinical Research Ethics Board of the University of British Columbia and BC Cancer Agency. Tissue Microarrays and IHC The Vancouver Hospital ER laboratory retained single archival blocks from each patient. This material had been frozen before neutral buffered formalin fixation. Slides from these blocks stained with hematoxylin and eosin were reviewed by two pathologists to identify areas of invasive breast carcinoma. Tissue microarrays (TMAs) were constructed as described 17,18 (details reproduced at papers/er.php). Using one core per patient, 17 TMA blocks were required. Thin sections (4 m) were immunostained using DakoCytomation EnVision and System-HRP (Dako Corporation, Carpinteria, CA) in a two-step technique. Slides were deparaffinized with xylene and rehydrated through three alcohol changes. Endogenous peroxidase activity was quenched by incubating 5 minutes with 0.03% hydrogen peroxide/sodium azide. Slides were then incubated with one of two primary anti-er antibodies, 1D5 (1:100; Dako Corp, Carpinteria, CA) or SP1 (1:250; LabVision, Fremont, CA), followed by the peroxidase-labeled polymer, in a Tris-HCl buffer containing stabilizing protein and an antimicrobial agent, using sequential 30-minute incubations. Staining was completed by a 10-minute incubation with 3,3 -diaminobenzidine plus substrate-chromogen. Primary antibody was omitted in negative controls. External positive controls were slides from breast cancers with previously documented ER expression. A previous study using a duplicate-redundancy 431-case TMA demonstrated 96% agreement between duplicate cores, for both 1D5 and SP1 9 (Tables A1 and A2). Stained TMA slides were digitally scanned and linked to a relational database, 19 and are available for review ( web/viewer.php; username, ersp11d5; password, er4150). ER Scoring System TMAs were scored visually by two pathologists (D.T., A.M.G.) for percentage of tumor cell nuclear positivity, and scored as negative ( 1%); positive, 1 (1% to 25%); positive, 2 (25% to 75%); or positive, 3 ( 75%). For most analyses, IHC scores are dichotomized at 1 ER positive. 20 Pathologists were blinded to clinical outcomes. Statistical Analysis Statistical analysis was performed using SPSS software version 13.0 (SPSS Inc, Chicago, IL) and R ( In univariate analyses, Overall survival (OS), breast cancer specific survival (BCSS), and relapse-free survival (RFS) were estimated using Kaplan-Meier 21 curves, and survival differences determined by log-rank tests. 22,23 A trend test was used when three or more ordered groups were compared. For BCSS, survival time was censored at death if the cause was not breast cancer, or if the patient was still alive at the end of the study. Six patients with unknown cause of death were excluded from BCSS analysis. For RFS, survival time was also censored at death if the cause was not breast cancer or the patient was alive without relapse at the end of the study. For OS, survival time was censored if the patient was still alive at the end of the study. Cox proportional hazards 22,24 models were used to calculate adjusted hazard ratios (HRs) accounting for covariates. Hypothesis testing was performed using Wald s statistic. Smoothed plots of weighted Schoenfeld residuals were used to test proportional hazard assumptions statistics and Kendall s -b 27 tests were used to measure agreement between the two ER immunostains and DCC assay, and correlation of ER status to pathologic variables. Differences involving pathologic factors were compared using Pearson s 28 and Mann-Whitney U 29 for categoric and continuous variables, respectively. All statistical tests were two-sided and P.05 was considered significant. RESULTS Our previous study 9 found that to detect ER expression, SP1 is more sensitive than 1D5 on both whole sections (5.3% absolute increased sensitivity) and TMAs (6.6%), and suggested that SP1 is the better prognostic marker for breast cancer survival (Tables A1 and A2). In this study we assessed the value of SP1 on a population-based series of 4,150 invasive breast cancers; 48% of patients had tumor 2 cm, 51% had grade 3 tumor, and 44% had positive nodal status (Table A3). Among the 1,838 patients undergoing lumpectomy, 91% received radiation, whereas 29% of the 2,241 patients undergoing mastectomy received radiation. Forty-one percent of patients received no AST and 33% received tamoxifen-only AST. Comparison of ER Expression by SP1 and 1D5 With DCC and Clinicopathologic Characteristics Table 1 summarizes the IHC staining results for SP1 and 1D5 on TMAs. Figures 1A to 1C are from the same tumor sample that was negative for ER by 1D5 but strongly positive by SP1. This sample had an ER concentration of 48 fmol/mg by DCC. Figures 1D to 1F are from another tumor sample, which was moderately positive by 1D5 but strongly positive by SP1 (ER concentration, 174 fmol/mg). The number of interpretable samples for the two immunostains is slightly different due to occasional core dropout from TMAs during sectioning and staining. Overall, 69.5% of samples were positive by SP1 versus 63.1% positive by 1D5. In absolute terms, the SP1 antibody had 11% more strong 3 stains than 1D5, and 1D5 had 6.4% more negative stains than SP1. There is a strong positive correlation between the two antibodies (Kendall s -b, 0.790; P ). Among clinical tumor samples, 3,884 samples were originally tested by DCC assay and categorized into four groups 10,11 : negative ( 1 fmol/mg), low (2 to 9 fmol/mg), moderate (10 to 159 fmol/mg), and high ( 160 fmol/mg). Frequencies were 2.9% (111 of 3,884), 17.5% (678 of 3,884), 41.1% (1,596 of 3,884), and 38.6% (1,499 of 3,884), respectively. Values of DCC 10.0 fmol/mg were considered Table 1. Frequencies of ER Antibodies SP1 and ID5 Immunostaining Expression in 4,150 Breast Cancer Patient Samples Expression Level SP1 No. of Patients % 1D5 No. of Patients % Negative ( 1%) 1, , %-25% %-75% 1, , % 1, Uninterpretable/missing core Total 4, , Abbreviation: ER, estrogen receptor JOURNAL OF CLINICAL ONCOLOGY

3 Comparison of ER IHC Methods in Breast Cancer A D B E Fig 1. (A to C) Patient 953, an infiltrating ductal carcinoma: (A) hematoxylin and eosin (H and E) histology; (B) tumor cells completely negative for expression of estrogen receptor (ER) using 1D5 monoclonal antibody; (C) 3 immunostaining for ER expression using SP1 monoclonal antibody. (D to F) Patient 963, an infiltrating ductal carcinoma: (D) H and E histology; (E) 2 moderate immunostaining by 1D5; (F) 3 immunostaining for ER using SP1. C F positive (the clinical cut point). 11,12 ER status by SP1 (, 0.654; sensitivity, 86%; specificity, 92%; P ) agreed better with DCC than did 1D5 (, 0.536; sensitivity, 78%; specificity, 92%; P ; Table 2). A total of 4,105 samples were interpretable for both antibodies. SP1 stained 8% more positive samples that otherwise were negative by 1D5 (Table 3). For these 337 discrepant samples, 92% were DCC positive (median concentration, 67.0 fmol/mg). Among the 77 discrepant cases identified as negative by SP1 but positive by 1D5, 65% Table 2. Comparison of ER Status as Detected by SP1 and 1D5 Immunohistochemistry, Using DCC as the Gold Standard ER Concentration Detected by DCC (fmol/mg) ER Status Result Predictive Value Detected by SP1 SP1 negative Negative, 62% SP1 positive 64 2,628 Positive, 98% Specificity, 92% Sensitivity, 86% Detected by 1D5 1D5 negative Negative, 51% 1D5 positive Positive, 97% Specificity, 92% Sensitivity, 78% Abbreviations: ER, estrogen receptor; DCC, dextran-coated charcoal. Table 3. Frequencies of SP1 and 1D5 Discrepancies Among Patient Samples Interpretable for Both SP1 and 1D5 Antibodies Negative SP1 Positive Total 1D5 Negative 1, ,513 Positive 77 2,515 2,592 Total 1,253 2,852 4,105 NOTE. SP1 identifies reclassifies 8.2% (337 of 4,105) positive of patient samples that would have been considered negative by 1D5, and 1.9% (77 of 4,105) negative of patient samples considered positive by 1D

4 Cheang et al were DCC positive (median concentration, 34.0 fmol/mg). The SP1-positive/1D5-negative discordant samples had significantly higher ER concentrations by DCC (P.021) and significantly lower tumor grades (P.022) than the SP1-negative/1D5-positive discordant samples; there was no significant difference in tumor size (P.110). Among the SP1-negative/1D5-negative group, 64% were DCC negative; among the SP1-positive/1D5-positive group, 98% were DCC positive. SP1-positive, 1D5-positive, and DCC-positive status all correlate with older age at diagnosis (Kendall s -b, [P ], [P ], and [P ], respectively), but correlate inversely with grade (Kendall s -b, [P ], [P ], and [P ]) and tumor size (Kendall s -b, [P ], [P ], and [P ]). Prognostic Values of SP1-, 1D5-, and DCC-Positive Expression To assess the prognostic value of ER in a general population, we examined SP1, 1D5, and DCC in the entire cohort. Both immunostains and DCC identified ER-positive patients as having an improved BCSS. DCC assay demonstrates the most significant and strongest linear trend for expression: the higher expression, the better BCSS (Fig 2A [SP1], P ; Fig 2B [1D5], P ; and Fig 2C [DCC], P ). A similarly significant trend is observed for RFS (SP1, P ; 1D5, P ; and DCC, P ) but not OS (SP1, P.132; 1D5, P.511; and DCC, P.154). The 5- and 10-year survival probabilities of binarized ER status from Kaplan and Meier analyses are listed in Table A4 (online only). The increased survival for ER-positive patients starts to attenuate after 5 years. The same phenomenon is seen with all three detection methods. AST for this cohort was prescribed according to guidelines based on age, tumor size, LVI, nodal status, and DCC-determined ER level. 16 High risk was defined as node positive, or if node negative, the presence of LVI or tumor more than 2 cm and ER negative (DCC 10 fmol/mg). Patients deemed low risk at the time of diagnosis were not given any AST; they had a better outcome (10-year BCSS, 82%; 95% CI, 80% to 84%) than patients receiving AST such as the tamoxifen group (10-year BCSS 69%; 95% CI, 66% to 71%; P ). Therefore, survival analyses based on ER status were done separately in these two subgroups. For the pure prognostic subset of patients receiving no AST, SP1-positive status was associated with 14% absolute increased BCSS (P ) at 10 years, 1D5-positive status was associated with 9% (P ), and DCC-positive status was associated with 19% (P ; Table A4). For RFS, SP1-positive and DCCpositive status were increased significantly by 6% (P.002) and 13% (P ) at 10 years. 1D5-positive status showed 3% increased 10-year RFS (P.06). Among 1,377 patients receiving tamoxifen as their only AST, only 84 were ER negative by DCC. SP1, 1D5, and DCC all identified ER-positive patients as having better outcomes than ER-negative patients. The absolute increased 10-year BCSS for SP1-, 1D5-, and DCCpositive status are 14% (P ), 10% (P ), and 24% (P ); the increased 10-year RFS for SP1-, 1D5-, and DCC-positive status are 10% (P ), 7% (P.001), and 18% (P ), respectively (Table A4). Concordant and Discordant Cases Between SP1 and 1D5 As expected, patients with SP1/1D5 double-positive status have better BCSS (HR, 0.667; 95% CI, to 0.753) compared with patients with SP1/1D5 double-negative status (P ; Fig 3). The SP1-positive/1D5-negative group has a HR of (95% CI, to 0.989; P.044) compared with the SP1-negative/ 1D5-positive group. Importantly, there are no significant survival differences between the SP1-positive/1D5-positive and SP1-positive/ 1D5-negative patients (P.408). The SP1-negative/1D5-positive group does not have significantly different survival from the SP1- negative/1d5-negative group (HR, 0.93; 95% CI, 0.63 to 1.36; P.698) but seems inferior to the SP1-positive group (HR, 1.45; 95% CI, 0.99 to 2.11; P.055). In relapse-free survival analysis, the SP1-negative/1D5-positive patients have a similar hazard compared with the SP1-negative/1D5-negative group, with a HR of (95% CI, to 1.38), whereas SP1-positive/1D5-negative patients have relapse-free hazard similar to that of SP1-positive/ 1D5-positive patients (HR, 1.07; 95% CI, to 1.28; (Fig A1, online only). The same pattern in BCSS and RFS is seen in both the subsets of patients not treated with AST or those treated only with tamoxifen (data not shown). These results also support that SP1 antibody is a better prognostic marker than the commonly used ER antibody 1D5. Multivariate Analysis Smoothed, rescaled Schoenfeld residuals plots were used to test proportional hazards assumptions. All covariates followed proportional hazards, except ER status, which varied slightly during the long period of follow-up. The hazard rate of breast cancer death and relapse is different in the first 5 years, consistent with data reported by Hess et al. 30 Table A5 summarizes the adjusted HRs of ER-positive status detected by the two immunostains and DCC; the reported values were determined from Cox regression models including age, tumor size, grade, LVI, and nodal status as covariates for BCSS and RFS (Table A5). The results show that SP1, 1D5, and DCC all work efficiently as independent prognostic factors for BCSS and RFS in a general population after adjusting for the listed clinicopathologic prognosticators. However, SP1, 1D5, or DCC did not remain significant among the low-risk patients receiving no AST. For patients receiving tamoxifen as their only AST, SP1, 1D5, and DCC each retained significance when added individually to a model including the same clinical parameters listed above as covariates. The BCSS HRs for SP1-positive, 1D5-positive, and DCC-positive status were (95% CI, to 0.811; P ), (95% CI, to 0.870; P ), and (95% CI, to 0.935; P ), respectively. The RFS HRs for SP1-positive, 1D5- positive, and DCC-positive status were (95% CI, to 0.864; P ), (95% CI, to 0.921; P ), and (95% CI, to 1.03; P.068), respectively. To test which detection method was more significantly associated with prognosis, a Cox regression model with age, tumor size, grade, LVI, and nodal status was fitted with each of SP1, 1D5, and DCC. SP1 was the most significant prognostic factor among the three ER detection methods, both in BCSS (HR, 0.664; 95% CI, to 0.852; P ) and RFS (HR, 0.699; 95% CI, to 0.888; P ) in this subgroup. The HRs of the clinicopathologic covariates are listed in Tables 4 and JOURNAL OF CLINICAL ONCOLOGY

5 Comparison of ER IHC Methods in Breast Cancer A Cumulative Breast Cancer Specific Survival SP1+ (> 75%) n = 1,228 SP1+ (25%-75%) n = 1,270 SP1- (< 1%) n = 1,255 SP1+ (1%-25%) n = 357 Cumulative Breast Cancer Specific Survival SP1+/1D5- n = 337 SP1-/1D5- n = 1,177 SP1+/1D5+ n = 2,535 SP1-/1D5+ n = 77 B Cumulative Breast Cancer Specific Survival C Cumulative Breast Cancer Specific Survival Log-rank test for linear trend (P = 4.78 x ) Total Follow-Up (years) SP1- (< 1% tumor nuclei) 10-year BCSS 65% (95% CI, 62 to 68) SP1+ (1%-25% tumor nuclei) 10-year BCSS 73% (95% CI, 68 to 78) SP1+ (25%-75% tumor nuclei) 10-year BCSS 75% (95% CI, 72 to 77) SP1+ (> 75% tumor nuclei) 10-year BCSS 79% (95% CI, 77 to 81) 1D5+ (> 75%) n = 772 1D5 - (< 1%) n = 1,520 1D5 + (1%-25%) n = 408 Log-rank test for linear trend (P = 1.65 x 10-7 ) Total Follow-Up (years) 1D5 + (25%-75%) n = 1,418 1D5- (< 1% tumor nuclei) 10-year BCSS 68% (95% CI, 65 to 70) 1D5+ (1%-25% tumor nuclei) 10-year BCSS 72% (95% CI, 67 to 76) 1D5+ (25%-75% tumor nuclei) 10-year BCSS 76% (95% CI, 74 to 79) 1D5+ (> 75% tumor nuclei) 10-year BCSS 78% (95% CI, 75 to 81) ER 160 fmol/mg n = 1,496 n = 1,593 ER fmol/mg ER 1 fmol/mg n = 111 Log-rank test for linear trend (P = 6.08 x ) Total Follow-Up (years) ER 1 fmol/mg 10-year BCSS 54% (95% CI, 44 to 63) ER 2-9 fmol/mg 10-year BCSS 62% (95% CI, 58 to 65) ER fmol/mg 10-year BCSS 72% (95% CI, 70 to 75) ER 160 fmol/mg 10-year BCSS 79% (95% CI, 77 to 81) ER 2-9 fmol/mg n = 678 Fig 2. Breast cancer specific survival (BCSS) for (A) SP1, (B) 1D5, and (C) dextran charcoal ligand-binding assays. The 10-year BCSSs are labeled accordingly. The linear trend across semiquantitative factor levels for survival differences was tested by log-rank. ER, estrogen receptor. Log-rank P = 9.9 x Total Follow-Up (years) SP1+/1D5+ 10-year BCSS: 76% (95% CI, 75 to 78) SP1-/1D5-10-year BCSS: 65% (95% CI, 62 to 67) SP1+/1D5-10-year BCSS: 78% (95% CI, 73 to 82) SP1-/1D5+ 10-year BCSS: 66% (95% CI, 54 to 76) Fig 3. Breast cancer specific survival (BCSS) of SP1 and 1D5 concordant and discordant cases (total, n 4,128). The 10-year BCSS estimates are labeled accordingly. The SP1-negative/1D5-positive group has a worse BCSS survival compared with the expected outcome from the rest of ER-positive patients. DISCUSSION Using population-based TMAs of more than 4,000 patients with longterm follow-up, this study not only validates the prognostic value of ER immunostaining in breast carcinomas with and without adjuvant tamoxifen, but also demonstrates the superiority of the SP1 rabbit monoclonal antibody to the standard 1D5 mouse monoclonal, both in agreement with DCC assay and in correlation with outcome. ER is a well-established determinant of survival and correlate of other clinicopathologic variables. 31 Accordingly, the vast majority of our analyses are confirmatory validations rather than tests of hypotheses, and a Bonferroni correction would be inappropriate. Because of reduced cost, shorter turnaround time, morphologic correlation, and ease of specimen handling, IHC testing for ER has largely replaced DCC assays 32 ; so high-quality antibodies play an important role in clinical decision making. Developed against an N-terminal epitope of ER-alpha, the mouse monoclonal antibody 1D5 is currently in wide use. 20 Another mouse monoclonal, 6F11, has similar sensitivity. 33,34 Previous studies using limited numbers of patients without outcome data suggested superior clinical sensitivity of the recently developed rabbit monoclonal antibody SP1, recognizing the ER-alpha C-terminal portion. 7 We show that SP1 is superior to 1D5 in identifying ER-positive patients who have a good prognosis, in the whole cohort (representing a British Columbia population), in patients receiving no AST (a pure prognostic group), and in patients receiving only tamoxifen AST. Our data suggest that 1D5 fails to identify some ER-positive patients who would benefit from adjuvant tamoxifen, thus potentially denying a well-tolerated, efficacious treatment to approximately 6% of breast cancer patients. SP1 more closely approximates the prognostic value of DCC assay than does 1D5. This suggests that IHC using SP1 is a better substitute for the previous gold standard in a clinical

6 Cheang et al Table 4. Cox Proportional Hazard Regression Analysis to Test the Best ER Detection Methods (SP1, 1D5, and DCC) on Patients Receiving Tamoxifen Only as Their AST: BCSS Characteristic BCSS (n 1,150) HR 95% CI P Age at diagnosis, years v to v to v to Grade II v I to III v I to Tumor size, cm 2-5 v to v to LVI Positive v negative to Nodal status Positive v negative to ER status by SP1 Positive v negative to NOTE. Age at diagnosis, grade, tumor size, LVI, and nodal status were included as covariates. Patient samples with missing values in any of the covariates or ER status were excluded in the analysis. Hazard estimates were not computed for the insignificant test variables. Abbreviations: ER, estrogen receptor; DCC, dextran-coated charcoal; AST, adjuvant systemic therapy; BCSS, breast cancer specific survival; HR, adjusted hazard ratio; LVI, lymphovascular invasion. In a model including SP1, 1D5, and DCC, SP1 is selected as the most significant contributor to BCSS (1D5, P.267; DCC, P.372). In a model including 1D5 as the only ER measure, 1D5-positive HR, (0.557 to 0.877), P In a model including DCC as the only ER measure, DCC-positive HR, (0.450 to 0.934), P Table 5. Cox Proportional Hazard Regression Analysis to Test the Best ER Detection Methods (SP1, 1D5, and DCC) on Patients Receiving Tamoxifen Only As Their AST: RFS Characteristic RFS (n 1,104) HR 95% CI P Age at diagnosis, years v to v to v to Grade II v I to III v I to Tumor size, cm 2-5 v to v to LVI Positive v negative to Nodal status Positive v negative to ER status by SP1 Positive v negative to NOTE. Age at diagnosis, grade, tumor size, LVI, and nodal status were included as covariates. Patient samples with missing values in any of the covariates or ER status were excluded in the analysis. Hazard estimates were not computed for the insignificant test variables. Abbreviations: ER, estrogen receptor; DCC, dextran-coated charcoal; AST, adjuvant systemic therapy; RFS, relapse-free survival; HR, adjusted hazard ratio; LVI, lymphovascular invasion; BCSS, breast cancer specific survival. In a model including SP1, 1D5, and DCC, SP1 is selected as the most significant contributor to BCSS (1D5, P.555; DCC, P.577). In a model including 1D5 as the only ER measure, 1D5-positive HR, ( ), P In a model including DCC as the only ER measure, DCC-positive HR, ( ), P diagnostic setting. Although IHC assays in general can suffer from interlaboratory variability, ER IHC has been shown to be robust and reproducible. 35 In this study, ER expression correlates with substantially greater improvements in disease-specific and relapse-free survival among tamoxifen-treated patients than among patients receiving no AST. In the latter group, although ER-positive patients (detected by any method) had better survival probabilities, their survival advantage slowly decreased and eventually crossed at 18 years (Table A4 and Fig A2, online only). This suggests some ER-positive patients have slow growing but high-risk tumors not related to tamoxifen resistance. Quantitative DCC results showed the best positive linear trend for better survival, and DCC is better than semiquantitative visual IHC to identify the dose effect of ER on outcome. Quantitative image analysis may be necessary for IHC techniques to match DCC assay in this regard. 32,35,36 Because of the exceptionally large study size, TMAs were constructed of single cores. Previous data from our laboratory found 96% agreement between duplicate cores for SP1 and for 1D5 immunostains 9 (Table A1). The almost five-fold difference in the numbers of SP1-positive/1D5-negative versus SP1-negative/1D5-positive discordant results in the current larger study argues strongly that the SP1- positive/1d5-negative patients (illustrated in Fig 1) do not merely represent false-negative 1D5 results. Because the applicability of these results to whole tissue sections might be questioned, we performed studies comparing SP1 and 1D5 in an unselected multi-institutional series of 121 whole-section 9 breast cancer specimens, and the absolute ER positivity rate was still 5.3% higher for SP1 compared with 1D5. This study also tested the two antibodies on a single-institution, 431-patient, formalin-fixed breast cancer TMA independent of the one presented here; as might be expected, the absolute rates of ER positivity were found to be higher in whole sections than in tissue microarrays (SP1, 83.5% [whole section] v 79.3% [TMA]; 1D5, 78.2% [whole section] v 72.7% [TMA]; Table A1), but importantly, the relative increase in sensitivity of SP1 versus 1D5 was maintained in both whole sections (5.3%) and the TMA format (6.6%), and agrees with the results presented here on 4,150 patients (6.4%). Although it is difficult to compare absolute rates because the contributing patient populations were different, in this 4,150-patient series, both antibodies did show lower absolute rates of positivity, which might reflect the fact that the source tissues (as per DCC protocol) were frozen before fixation. The fact that DCC assay was positive in 36% of the tumors, which were negative by both 1D5 and SP1 IHC, supports loss of ER immunoreactivity in tissues frozen before fixation. We provide evidence that the new rabbit monoclonal antibody SP1 represents an improved standard for IHC ER assessment in breast cancer. Recent studies have made accurate assessment of ER status even more critical, given its implications for predicting response to systemic therapies JOURNAL OF CLINICAL ONCOLOGY

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For a detailed description of the disclosure categories, or for more information about ASCO s conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Authors Employment Leadership Consultant Stock Honoraria Research Funds Testimony Other C. Blake Gilks Aventis Pharma (now Sanofiaventis)

8 Cheang et al Author Contributions Conception and design: Maggie C.U. Cheang, Diana O. Treaba, David Huntsman, C. Blake Gilks, Torsten O. Nielsen, Allen M. Gown Financial support: Karen A. Gelmon, Torsten O. Nielsen Administrative support: Ivo A. Olivotto, David Huntsman Provision of study materials or patients: Ivo A. Olivotto, Karen A. Gelmon, C. Blake Gilks, Torsten O. Nielsen Collection and assembly of data: Maggie C.U. Cheang, Diana O. Treaba, Caroline H. Speers, Ivo A. Olivotto, Lynn C. Goldstein, Karen Gelmon, David Huntsman, C. Blake Gilks, Torsten O. Nielsen, Allen M. Gown Data analysis and interpretation: Maggie C.U. Cheang, Diana O. Treaba, Caroline H. Speers, Ivo A. Olivotto, Chris D. Bajdik, Stephen K. Chia, Torsten O. Nielsen, Allen M. Gown Manuscript writing: Maggie C.U. Cheang, Diana O. Treaba, Ivo A. Olivotto, Chris D. Bajdik, Stephen K. Chia, Torsten O. Nielsen, Allen M. Gown Final approval of manuscript: Maggie C.U. Cheang, Diana O. Treaba, Caroline H. Speers, Ivo A. Olivotto, Chris D. Bajdik, Stephen K. Chia, Lynn C. Goldstein, Karen A. Gelmon, David Huntsman, C. Blake Gilks, Torsten O. Nielsen, Allen M. Gown 5644 JOURNAL OF CLINICAL ONCOLOGY